The ubiquitous, yet poorly understood, phenomenon of intrinsic disorder in proteins has wide-ranging implications for fundamental questions in molecular biology and the design of novel small molecule pharmaceuticals. Intrinsically disordered proteins inherently lack secondary and/or tertiary structure under physiological conditions;they couple folding with binding to their interaction partners with high specificity and, often, extraordinary versatility. The signatures of disorder are found at the heart of transcriptional regulation, protein interaction networks, and a number of diseases. New methods to target and manipulate these protein-protein interactions are urgently required. Understanding the mechanisms in detail, from selection or encounter through intermediates to the bound form, will lead to significant advances in the efficient design of small molecules to control these interactions. Additionally, resolving the principles governing the function of these small, highly specific proteins will further de novo design of peptides and mimics, a growing pharmaceutical aim. This work proposes a combination of NMR spectroscopy, molecular dynamics and molecular biology to gain unique structural insights into coupled folding and binding. Specifically, this work will utilize the complex of the phosphorylated kinase inducible transactivation domain (pKID) of CREB with the KIX domain of CBP as a model system to generate detailed views of the encounter complex and intermediate(s) along an induced folding pathway. Specifically, this proposal aims to (1) define the evolution of secondary structure as distinct from intermolecular interactions in the induced folding pathway, (2) separate the effect of fly-casting from electrostatic steering in docking, and (3) generate detailed models of the binding intermediate(s). For aim 1, the work will utilize relaxation dispersion NMR spectroscopy and the sensitivity of the carbonyl and alpha carbons to secondary structure to quantify the residue specific helical content in the intermediate(s). For aim 2, pKID constructs of varied lengths will be systematically mutated and measured by isothermal calorimetry to separate the effects of charge and construct length on binding thermodynamics. For aim 3, the work will utilize NMR-derived restraints to limit the torsion angles and relative positions in molecular dynamic simulations of the intermediate(s) in the binding pathway. The combination of these studies will significantly advance our understanding of the fundamental mechanisms and guiding principles in the association of intrinsically disordered proteins. The unique structural insights will greatly increase our ability to design small molecules and peptides to bind and disrupt protein-protein interactions. ) PUBLIC HEALTH RELEVANCE: Proteins that inherently lack ordered structure are central to both healthy biological functions and a number of diseases. Disordered proteins often form ordered structure only upon binding an interaction partner forming unique surfaces difficult to target with traditional pharmaceutical design methods. The proposed work will greatly advance our understanding of the mechanisms of disordered proteins as well as our ability to effectively design new pharmaceuticals to target and disrupt protein-protein interactions. )